Danielle Pesando

Preliminary survey of toxicity of the green alga Caulerpa taxifolia introduced into the Mediterranean

Caulerpa taxifolia (Vahl) C. Agardh (Ulvophyceae, Caulerpales) is an alga of tropical origin that was accidentally introduced into the Mediterranean sea in 1984, where this species can reach an abundance that has never been described in tropical endemic regions. It is known that caulerpacean algae can develop an efficient strategy against grazers consisting of the synthesis of repulsive of toxic secondary metabolites: we report here the first study of the toxicity of purified secondary metabolites and raw extracts fromC. taxifolia from the Mediterranean.Toxicity was evaluated on three models: mice (lethality), mammalian cells in culture (cytotoxicity) and sea urchin eggs (disturbance of cell proliferation). Aqueous extracts are only active on fibroblasts and mice. In the three toxicity models a seasonal variation of toxicity is observed for the crude methanol extract as well as a decrease of this activity whenC. taxifolia from the Mediterranean is kept in aquaria. Pure compounds exhibit different toxicity depending on the assay. 10,11-epoxycaulerpenyne is the most active substance on mice and fibroblasts whereas taxifolial A and D are inactive or only weakly toxic. Among the four tested compounds caulerpenyne, the major metabolite ofC. taxifolia, is the most active on sea urchin eggs. Caulerpenyne may therefore represent an ecological risk for microorganisms and the eggs of multicellular animals living close to this alga. The ecological impact of this toxicity on marine organisms and the interaction of this alga with the herbivorous fauna are discussed.

Biological targets of neurotoxic pesticides analysed by alteration of developmental events in the Mediterranean sea urchin, Paracentrotus lividus

Biological effects of neurotoxic insecticides widely used for agricultural purposes were studied using the early development of the Mediterranean sea urchin Paracentrotus lividus as a model. These compounds, dispersed as aerosols or powders in agricultural regions near to the coast, may affect the health of organisms in the marine environment. The biological effects of Basudin (an organophosphate compound containing 20% Diazinon), Diazinon (Dzn, a thionophosphate), Carbaryl and Pirimicarb (carbamates) on the early phases of sea urchin development were thus investigated. Morphological, biochemical, histochemical and immuno histochemical analyses were performed both during embryo and larval development. For the morphological effects on fertilisation and first cleavages, the effective concentration of insecticides was found to be 10(-4) M, while for further stages concentrations between 10(-5) and 10(-7) M were effective: 10(-3) M of any of these insecticides totally arrested development. During embryonic development, the treatment with organophosphates slowed the rate of early mitotic cycles down, affected nuclear and cytoskeletal status as well as DNA synthesis. From the gastrulation stage onwards, the main effects were exerted on the rate of primary mesenchyme cells migration, larval size, perioral arm length, and acetylcholinesterase activity distribution, thus deregulating the cholinergic system, which modulates cell-to-cell communication mediated by the signal molecule acetylcholine.

Effects of caulerpenyne, the major toxin from Caulerpa taxifolia on mechanisms related to sea urchin egg cleavage

Abstract Caulerpenyne (CYN), the major metabolite synthesized by the alga Caulerpa taxifolia (Vahl), inhibited the first cleavage of sea urchin eggs without affecting fertilization. The effect was dose-dependent with a half maximal dose of 33 μM. Blockage of cleavage was observed when the toxin was added within 40 min of insemination. A preliminary search for the cellular targets of this toxin showed that ionic signals involved in the cell dynamics are altered: caulerpenyne reduced the intracellular ATP-dependent Ca 2+ accumulation in a dose-dependent manner but did not provoke a release of sequestered Ca 2+ . This effect is similar to that of thapsigargin, a specific inhibitor of reticular Ca 2+ -ATPase. CYN had no effect on the incorporation of 35 S-methionine into proteins. 3 H-thymidine incorporation into DNA was inhibited by CYN in a dose-dependent manner: an effect well correlated with cell division kinetics. A CYN concentration of 30 μM, which delayed the first cleavage, inhibited overall protein phosphorylation but did not affect histone kinase phosphorylating activity. Thus, CYN appears to alter the main events of sea urchin egg cleavage, and may therefore constitute an ecological risk for microorganisms and eggs of pluricellular animals living close to these algae. Moreover, this compound is of potential pharmacological interest in view of its antiproliferative properties.

Toxic effects of Gymnodinium cf. mikimotoi unsaturated fatty acids to gametes and embryos of the sea urchin Paracentrotus lividus

Abstract The toxicity of the main Gymnodinium cf. mikimotoi polyunsaturated fatty acid, has been investigated using the sea urchin gamete and embryo bioassays. The 18:5n3 fatty acid delays or inhibits first cleavage of Paracentrotus lividus eggs and provokes abnormalities in the embryonic development. These effects were compared with those of other polyunsaturated fatty acids, 18:4n3, 20:5n3 and 22:6n3, which are also present in this alga. A classification of the different fatty acids, based on their effects on sea urchin egg cleavage and the determination of the half inhibiting concentrations (IC 50 ) is proposed.

Effects of tributyltin on Ca2+ homeostasis and mechanisms controlling cell cycling in sea urchin eggs

Abstract Tributyltin (TBT) is one of the widespread organotins in the marine environment: we have investigated its cellular targets in the eggs of the marine invertebrate sea urchin Paracentrotus lividus . TBT was used at concentrations ranging from 10 −9 to 10 −4 M. The lipophilic character of TBT makes that the magnitude of the effects varied according to the concentration of egg used. Concentrations of TBT being without effect on egg fertilisation inhibited first and second cleavage in a dose-dependent manner. Concentrations of TBT which block egg cleavage did not alter egg membrane permeabilities to Ca 2+ and Na + . TBT inhibited intracellular ATP-driven sequestration of Ca 2+ into reticular compartment but did not provoke Ca 2+ leakage from these compartment. Blockade of eggs cleavage by TBT is accompanied by an inhibition of protein synthesis ( 35 S-methionine incorporation) and DNA synthesis ( 3 H-thymidine incorporation). Protein phosphorylation ( 32 P incorporation) and histone H1 kinase activity were inhibited when cell cycling was arrested by organotin. Results are discussed in terms of concentration of TBT per egg protein in order to compare the particular sensitivity of the numerous cellular process affected by TBT.

MICROALGAE : A MODEL TO INVESTIGATE THE ECOTOXICITY OF THE GREEN ALGA CAULERPA TAXIFOLIA FROM THE MEDITERRANEAN SEA

Abstract The spectacular development of the green alga Caulerpa taxifolia (Vahl) C. Agardh, introduced in the Mediterranean in 1984, represents a biological pollution which threatens the biodiversity of the marine ecosystem. The weak pressure from grazers and the presence of repulsive secondary metabolites make the problem worse. Whereas the anti-appetant effect of these metabolites is well known, their role in competition between algae has not been extensively studied. Using a microalgal model representing the initial stage of the marine food chain, we studied the effects of C. taxifolia metabolites. We showed that organic extracts of C. taxifolia and caulerpenyne inhibit or delay the proliferation of several phytoplanktonic strains to various degrees. Seasonal variations of the toxicity were observed with a maximal effect in the summer. Experiments with Dunaliella minuta showed that caulerpenyne did not affect the protein content but strongly reduced that of the cell chlorophyll A.

EVALUATION OF THE TOXICOLOGICAL RISK TO HUMANS OF CAULERPENYNE USING HUMAN HEMATOPOIETIC PROGENITORS, MELANOCYTES, AND KERATINOCYTES IN CULTURE

The extensive growth of Caulerpa taxifolia in the Mediterranean sea produces important quantities of bioactive secondary metabolites unable to enter the food chain. The cytotoxic effects of caulerpenyne, the major secondary metabolite from C. taxifolia, was studied in different in vitro models: skin cells, primary cultures of melanocytes and keratinocytes, immortalized keratinocytes (HaCaT and HESV), and bone marrow cells (hematopoietic progenitors CFU-GM). Typical dose-response curves from neutral red uptake and MTT assays were recorded in all models with IC50 ranging from 6 to 24 microM. Hematopoietic progenitors were more sensitive to caulerpenyne than melanocyte and keratinocyte cell lines, which could be due to their higher proliferative rate. The distribution of aggregates in colonies, macroclusters, and microclusters of hematopoietic progenitors was also altered in the presence of caulerpenyne. From our evaluation of the caulerpenyne concentrations required to result in cellular toxicity, the risks of cutaneous and/or food intoxication to humans may be considered minimal.

Effect of caulerpenyne, a toxin extracted from Caulerpa taxifolia on mechanisms regulating intracellular pH in sea urchin eggs and sea bream hepatocytes.

The proliferation of the green marine alga Caulerpa taxifolia in the Mediterranean led us to investigate the toxic effects on marine organisms of caulerpenyne (Cyn), the major secondary metabolite synthesized by the alga. This study was performed on sea urchin eggs (Paracentrotus lividus) and isolated hepatocytes from the sea bream (Sparus aurata), in which accumulation of the toxins by metabolic processes may be of significance. Cyn provoked an acidification of seawater containing both unfertilized and fertilized eggs, as revealed by a titrable efflux of protons. The pHi in unfertilized eggs continuously increased in the presence of Cyn, whereas there was a biphasic response in both fertilized eggs and isolated hepatocytes, with a decrease of the pHi followed by recovery to the initial value. Cyn inhibited the accumulation of 14C-methylamine in acidic granules present in the cortical zone of sea urchin eggs. Dicyclocarbodiimide (DCCD), a well-known H(+)-ATPase inhibitor, provoked a similar inhibition. Both molecules increased pH in the acidic compartments of isolated bream hepatocytes. These results suggest that Cyn inhibits intracellular sequestration of protons and thus liberates protons into the cell cytoplasm from which they leak toward the extracellular medium.

Caulerpenyne interferes with microtubuledependent events during the first mitotic cycle of sea urchin eggs

Caulerpenyne (Cyn), the major secondary metabolite synthesized by the green alga Caulerpa taxifolia proliferating in the Mediterranean Sea, is a cytotoxic sesquiterpene. As this compound has an antiproliferative potency by inhibiting division of many types of cells, we examined the precise effects of Cyn during the early development of the sea urchin Paracentrotus lividus . Whereas Cyn (60 µM) had no effect on fertilization, it blocked the first cell division in the same manner whether added before or after fertilization, provided the drug was added before or during metaphase. Immunofluorescence localization revealed that Cyn had no effect on the microtubular sperm aster formation, pronuclei migration and fusion, chromosome condensation, nuclear envelope breakdown, and bipolar mitotic spindle assembly. However, mitosis was blocked in a metaphase-like stage at which most chromosomes were aligned at the equatorial plate, while a few of them had not even migrated towards the metaphase plate. When added after the metaphase-anaphase transition, the first division occurred normally but the second division was inhibited with the same phenotype as described above. We previously showed that Cyn did not affect protein synthesis or H1 kinase activation or deactivation (Pesando et al., 1996, Aquat. Toxicol. 35,139), but that it partially inhibited DNA synthesis. Our results establish that Cyn does not affect the microfilament-dependent processes of fertilization and cytokinesis and allows the beginning of mitosis, but prevents normal DNA replication and results in metaphase-like arrest of sea urchin embryos.

Age et croissance de l'oursin Paracentrotus lividus Lamarck, 1816 (Echinodermata-Echinoidea) dans le golfe de Tunis (Mediterranee)

Abstract Age and growth of Paracentrotus lividus Lamarck, 1816 (Echinodermata-Echinoidea) in the gulf of Tunis (Mediterranean Sea). The age of Paracentrotus lividus in the gulf of Tunis was evaluated by analysing the natural growth banding in the interambulacral plates from the oral part of the test. It appeared that growth rates increased in spring and summer, and decreased in autumn and winter. The maximum age and diameter were estimated to be 8 years and 53 mm respectively. Compared to populations of Paracentrotus lividus from NW Mediterranean and Atlantic, that of the gulf of Tunis presented a more rapid growth.